The mammalian target of rapamycin (mTOR) signaling network is a master regulator of essential cellular and developmental processes, including cell growth, proliferation, cellular differentiation, and metabolism. Dysregulation of mTOR signaling has been found in a wide range of human diseases such as cancer, metabolic diseases, and neurological disorders. The overall goal of the proposed studies is to understand mTOR complex 1 (mTORC1) signaling mechanisms in cell growth regulation, with a focus on the lipid second messenger phosphatidic acid (PA). Our previous work established PA and its biosynthetic enzyme phospholipase D (PLD) as critical mediators of mitogenic and amino acid activation of mTORC1. In the current grant cycle we have gained new insights into the mechanisms and upstream pathways in PA regulation of mTORC1. These recent discoveries have taken us into new directions of investigation with the promise of deeper molecular understanding of this complex signaling network. Combining well-established biochemical approaches with biophysical methods that we have recently developed, we will dissect the biochemical mechanisms of PA- mTORC1 signaling in the experimental system of mammalian cell culture. Our specific aims are to (1) investigate leucyl tRNA synthetase (LRS) as an amino acid sensor for the Vps34-PLD-mTORC1 pathway, (2) dissect the interaction between PA and mTOR using single-molecule assays, and (3) study novel mTOR- interacting proteins under the regulation of PA. Our biochemical and biophysical expertise and strong preliminary data put us in an ideal position to pursue the proposed studies. Accomplishing these aims will likely lead to novel mechanistic insights into mTORC1 signaling and regulation of cell growth. The new regulators and molecular interactions uncovered could be potentially explored in the future as therapeutic targets in diseases involving mTOR, such as cancer.